Experimental Study On The Effect And Mechanism Of Danhong Injection On Acute Myocardial Injury Subsequent To Severe Acute Pancreatitis In Rats

Part I To Set up the Model of Acute Myocardial Injury Subsequent toSevere Acute Pancreatitis in RatsObective: To set up the model of acute myocardial injury in rats with severeacute panctreatitis with a methods that was of operability, repeatability and good stability;and to observe the regularity of morphological changes of injured myocardium in theprocess of severe acute panctreatitis,as well as to observe the regularityof serum variationof CK-MB,cTnI in the process of severe acute panctreatitis. Methods:Sixtymale S-D ratswere divided into experimental group (SAP group,per ten rats for 4h,8h,12h time pointafter the model set up) and control group(normal group,thirty rats) according to the rulesof comparative experimental study.Those in group SAP received two injections of 3.2g/kgbody weight L-Arg( 25% of the concentration) i.p. at an interval of 1h. Per 10 rats in SAPgroup and control group were exsanguinated 28h,32h,36h after the second L-Arginjection. The specimen of pancreas were observed under microscope.The specimen ofhearts were observed under both microscope and electron microscope.The serum CK-MBand cTnI were detected at the same time.Results:1.Pathological changes of pancreas andmyocardium under light microscope:(1) pancreas in SAP group:Pancreatic interlobularseptum and interstitial became widened;pancreatic alveolar swelled,bleeded andnecrosed;pancreatic lobular structure destructed and necrosed in coagulation state;there still existed longly pancreatic acinar;inflammatory cell infiltrated pancreaticstructure;and the pathological changes were more severe as the process developed.Thepancreatic structure in control group were normal under light microscope.(2) myocardium in SAP group:Myocytes swelled;some myocytes collapsed and myocardial fiber structuredisappeared;some inflammatorycell infiltrated into myocardial fiber,and the pathologicalchanges were more severe as the process devepoped.The myocardical structure in controlgroup were normal under light microscope.2.Myocardium under electron microscope:(1) myocardium in SAP group:Myocytes,nucleus and mitochondria swelled;mitochondrial cristae disappeared;Sarcoplasmic reticulum expansed;some of themyofibril fractured;filament decomposed.The sub-cell structure of myocytes in controlgroup under electron microscope were normal,the structure of sarcolemma and organellewere normal.3.The changes of serum CK-MB and cTnI:The serum CK-MB and cTnI ingroup SAP went up at 4h time point,went up higher at 8h,and reached to the peakat12h.The difference between each time point in group SAPwere significant(P<0.05).Theserum CK-MB and cTnI in control group were in normal scope.The difference betweenthe same time point in group SAP and control group were significant(P<0.05).Conclusion:S-D rats received two injections of 3.2g/kg body weightL-Arg( 25% of the concentration) i.p. at an interval of 1h can be induced SAPmodels.Thismethods was of operability, repeatabilityand good stability.Acute myocardial injury(AMI)in rats with severe acute panctreatitis were observed at the early stage ofprocess.Pathological changes of myocardium,serum CK-MBand cTnI were observed atthe early stage of process,too.The changes of cardiac morphology and serumbiochemistry were correlated with the severity of SAP.It suggested that acute myocardialinjury(AMI) is a severe complication in the prossess of SAP.Part II The Mechanism of ET,NO and Adhesion molecule in AcuteMyocardial Injury Subsequent to Severe Acute Pancreatitis in RatsObjective: To investigate the mechanism of microcirculation dysfunction in AMIassociated with SAP in rats. Materials and Methods: the 60 male SD rats were randomlydivided into SAP model group (n = 30) and control group (n = 30), according to the first part of the method of manufacturing SAP model. Myocardial tissue were extracted at 4h,8h, 12h time point after modeled. RT-FQ-PCR (Real-time fluorescence quantitativepolymerase chain reaction) were used to detect the expression of those gene that wererelated closely to the microcirculation of the myocardial tissue(including ET-1,iNOS,eNOS,P-selectin, ICAM-1 and VCAM-1 gene). Simultaneous,serum concentration ofthose genetic expression product were detected after the aortic blood were centrifugated.Results: 1. The expression of those gene that were closelyrelated to microcirculation:Thegenetic expression of ET-1,eNOS,iNOS,ICAM-1,P-selectin were at lower levels incontrol group,and there were no significant difference among the 4h,8h and 12h timepoints in control group. The expression of those gene in SAP group at 4h,8h and 12htimepoints were significantly higher than that in control group of the same time points. 2.The serum concentration changes of Vasoactive mediators and adhesion molecules:Theserum concentration of ET-1,P-selectin,IVAM-1,ICAM-1 and the activity of iNOS,eNOS were at lower levels in control group. In SAP group,the serum concentration ofET-1,P-selectin,IVAM-1,ICAM-1 at 4h,8h and 12h time points were significantlyhigherthan that in control group of the same time points(P<0.05). The activity of iNOS andeNOS began to rise at 4h time point,8h time point,and reach to peak levels at 12h timepoint. Conclusion: Those Vasoactive mediators (ET, NO) and adhesion molecules,(P-selectin,ICAM-1,VCAM-1) that were closely related to microcirculation playimportant roles in acute myocardial injury induced by severe acute pancreatitis in rats. Themicrocirculatory dysfunction is one of important mechanisms that lead to SAP-associatedmyocardial injury.Part III The Protecting Effects of Danhong Injection on AcuteMyocardial Injury Subsequent to Severe Acute Pancreatitis in RatsObjective:To investigate the effects of Danhong injection on acute myocardialinjury subsequence to SAP inrats. Materials and Methods:Sixty male SD rats wererandomlydivided into SAP model group (n = 30) and Danhong injection treated group (n = 30), according to the first part of the method of manufacturing SAP model. At 4h, 8h 12htime point after SAP models built ,myocardial tissue in rats of SAP group and treatmentgroup were extracted. RT-FQ-PCR were used to detect the expression of those gene thatwere closely related to the microcirculation of the myocardial tissue(including ET-1,iNOS,eNOS,P-selectin, ICAM-1 and VCAM-1 gene). Simultaneous,serum CK-MB,cTnI and serum concentration of those genetic expression product were detected after theaortic blood were centrifugated. The specimen of myocardial tissue extracted from bothSAP group and treatment group were observed under light microscope. Results:1. Theexpression of those gene that were closely related to the microcirculation of the myocardialtissue(including ET-1,iNOS,eNOS,P-selectin, ICAM-1 and VCAM-1 gene): Theexpression of ET-1,iNOS,P-selectin, ICAM-1 and VCAM-1 gene (RQ value) in thetreatment group at 4h,8h and 12h time point were significantly lower than that in SAPgroup of the same time points. Meanwhile,the expression of eNOS gene in the treatmentgroup at 12h time point was significantly higher than that in SAP group of the same timepoint. 2. The changes of Serum CK-MB, cTnI, ET-1, P-selectin, IVAM-1, ICAM-1concentration and iNOS, eNOS activity: The serum concentration of CK-MB, cTnI, ET-1,P-selectin, IVAM-1, ICAM-1 and serum iNOS, eNOS activity in rats of SAP were at highlevels. The serum concentration of CK-MB, cTnI, ET-1, P-selectin, IVAM-1, ICAM-1 andserum iNOS activity in rats of treatment group were lower than the SAP group at the sametime point(P<0.05). Meanwhile,serum eNOS activity in rats of treatment group washigherr than that of SAP group at the same time point 3. Pathological changes ofmyocardial tissue under light microscope: the myocardial tissue in rats of control groupwere in normal morphological state. In SAP group,some myocardial cells were swollen,other myocardial cells were collapsed, and the myocardial fiber structure were disappeared.Some inflammatory cell infiltrated into myocardial fiber,and the pathological changeswere more severe as the course aggravated.In treatment group,the arrangement of themyocardial cells were mainly in normal state, the myocardial interstitial spaces wereedema, some muscle fiber structure were disappeared and disordered, fewer inflammatory cell infiltrated into myocardial fibers . Conclusion:The Danhong injection could reducethe expression of those genes(ET, P-selectin, ICAM-1, VCAM-1, iNOS)as well asincrease the expression of eNOS genes in myocardial tissue,and could reduce serumconcentrations of ET, P-selectin, ICAM-1, VCAM-1 and serum activity of iNOS as well asincrease serum activity of eNOS,too. Dan Hong injection can better myocardial injurysubsequent to SAP.